1 Department of Animal Reproduction and Veterinary Radiology of the School of Veterinary Medicine and Animals Husbandry (FMVZ), São Paulo State University (UNESP) at Botucatu, State of São Paulo, Brazil; 2 School of Medicine of Botucatu (FMB), São Paulo State University (UNESP) at Botucatu, State of São Paulo, Brazil; 3 Department of Clinical Analysis, School of Pharmaceutical Sciences of Araraquara, São Paulo State University (UNESP) at Araraquara, State of São Paulo, Brazil; 4 Department of Clinicsof the School of Veterinary Medicine and Animal Husbandry (FMVZ), São Paulo State University (UNESP) at Botucatu, State of São Paulo, Brazil; 5 Department of Tropical Diseases of the School of Medicine of Botucatu (FMB), São Paulo State University (UNESP) at Botucatu, State of São Paulo, Brazil; 6 Center for the Study of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP) at Botucatu, State of São Paulo, Brazil.

ABSTRACT: Sequelae due to testicular biopsy such as hemorrhage, adhesion and fibrosis may be limiting factors to the use of this surgical procedure. Fibrin glue (FG) derived from snake venom was used to minimize these sequelae, as well as to evaluate its healing property in tunica vaginalis and scrotal skin of rams. Applicability of fibrin glue derived from snake venom was tested in different tissues of other animals such as in sciatic nerve and colon of rats and skin of rabbits. In the present study,30 healthy adult rams were used. They were divided into 3 groups of 10 animals each as follows: G1: fibrin glue group (application of fibrin glue on puncture sites and skin incisions after bilateral testicular biopsy with a Tru-Cut needle); G2: swab/nylon group (hemostasis by compression with a swab on puncture sites and skin suturing with nylon after biopsy) and G3: control group (the animals were not subjected either to biopsy or to surgery). On the 20 day after biopsy, the presence of adhesion strands between the sites of skin incision and testicle was evaluated by palpation. Adhesion strands were found in three testicles (15%) in G1 and in two testicles (10%) in G2. One hundred days after biopsy, orchiectomy was carried out and the material collected was assessed for subcutaneous (SC) and/or tunica vaginalis adhesions. G3 did not present any abnormality. Groups G1 and G2 presented four testicles each (20%) with adhesion betweenthe tunics at biopsy site. On the other hand, subcutaneous adhesions were found once (5%) in G1 and three times (15%) in G2. Fibrin glue showed to be of easy application, required short postoperative monitoring, presented fast and good-quality healing property and tended to reduce formation of subcutaneous adhesion. KEY WORDS: Fibrin tecidual adhesive, snake venoms, biopsy, testicle, ovine.

INTRODUCTION

Testicular biopsy is a useful technique for fertility investigation. It has also been a helpful tool in the diagnosis of germinal cell aplasia, neoplasias(18), immune-mediated infertility(19), sex chromosomal anomalies and disorders of sex differentiation(27), in addition to permitting the assessment of an individual's over-all daily sperm production(12). However, hemorrhage, hematomas, adhesion and fibrosis(3,6,14,24) are complications that may occur after testicular biopsy and may limit its use.

Surgeries using fibrinogen and fibrin with hemostatic property have been performed since the beginning of this century. In 1909, Bergel(1) used fibrin powder in a blood vessel wall, and in 1915 Grey(9) used fibrin in cerebral surgeries. Since then, several studies have been conducted in an attempt to standardize a product with adhesive and hemostatic properties. In 1940, Young and Medawar(28) obtained good results when attempting to unite the sciatic nerve of rabbits. However, in 1944 Cronkite et al.(4) were unsuccessful in the use of fibrinogen and thrombin in skin grafts in humans. According to Lerner and Binur(17), these and other studies were not successful, probably due to the use of low concentrations of fibrinogen. As a result, the use of this biological adhesive did not become popular until the 1970s.

With the development of microsurgery and the greater availability of cryoprecipitate fibrinogen concentrate and other constituents, research was resumed in the 1970s with the revival of the so-called fibrin glue with higher concentration of fibrinogen in microanastomoses of nerves and vessels(17).

Posteriorly, fibrin glue was used to achieve hemostasis of liver and splenic injuries, mainly in patients with coagulation disorders due to its local homeostatic action, regardless of the individual's general condition(13). Fibrin glue was also used in canine liver biopsies with homologous fibrinogen(26), in neuro, ophthalmic(8), plastic, cardiac, lymphatic, genito-urinary, orthopedic surgeries and others as mentioned by Lerner and Binur(17).

De Virgilio et al.(5) and Sheppard et al.(21) working in the abdominal cavity of rats found that aside from its adhesive and hemostatic properties, fibrin glue was effective in inhibiting postoperative adhesion formation. According to Byrne et al.(2) and Lasa Jr. et al.(15), fibrin glue plays an important role in wound healing in rats. Gibble and Ness(7) reported the use of fibrin glue in human skin healing.

Fibrin glue results from a complex of fibrinogen concentrate and bovine thrombin reconstituted in a solution of calcium chloride. Fibrinogen complex also contains fibronectin, coagulation factors VIII and XIII, von Willebrand's factor and plasminogen. When mixed together, the components coagulate on tissue surface, resembling the last step of the coagulation process(13,15).

Researchers from the Center for the Study of Venoms and Venomous Animals - CEVAP (10,25) of São Paulo State University - UNESP standardized another type of fibrin glue derived from snake venom. This new glue consists of a cryoprecipitate containing human, bovine, bubaline or equine fibrinogen and a "thrombin-like" fraction of snake venom. This glue is different from the traditional glues since it uses animal instead of human fibrinogen and it also substitutes bovine thrombin for a" thrombin-like" enzyme extracted from Crotalus durissus terrificus venom which was isolated by Raw et al.(20). The "thrombin-like" enzyme is much more potent than bovine thrombin in the conversion of fibrinogen into fibrin(23) .The utilization of this glue has the advantage of permitting the use of homologous, autologous and heterologous fibrinogen, according to necessity.

Applicability of fibrin glue derived from snake venom was tested and approved in different animal tissues.

Viterbo et al.(25) and Iuan et al.(11) tested the fibrin glue derived from snake venom in the repair of sciatic nerve of Wistar rats and observed its good hemostatic and adhesive properties, as well as a satisfactory regeneration of the sealed nerves. Viterbo et al.(25) used bubaline, equine, bovine and human fibrinogen. These authors concluded that fibrin glue derived from snake venom and bubaline fibrinogen showed to be a major alternative for the repair of peripheral nerves.

Leite et al.(16) tested the fibrin glue derived from snake venom in anastomoses of colon of Wistar rats with good results. The glue they used was made up of snake venom fraction and bubaline fibrionogen.

Stolf et al.(22) evaluated this fibrin glue in skin of rabbits subjected to elliptical skin incision on the trunk. The results obtained suggested that the glue acts efficiently on the healing process due to an increase of a fibrin network.

The objective of the present study was to observe the results obtained with the use of fibrin glue derived from snake venom (with homologous fibrinogen) in testicular biopsy of rams. These results were observed with regard to hemorrhage control, prevention of testicular adhesions, effect of the glue as a sealing technique and its healing property in the testicle, tunica vaginalis and skin.

MATERIALS AND METHODS

STANDARDIZATION AND PREPARATION OF FIBRIN GLUE DERIVED FROM SNAKE VENOM: The isolation of the "thrombin-like" enzyme from Crotalus durissus terrificus venom and the preparation of the cryoprecipitate containing fibrinogen are described by Iuan et al.(11) and Thomazini et al.(23).

The cryoprecipitate from ovine contains an average fibrinogen concentration of 267.70 mg% in addition to factors VIIIc, XIII, von Willebrand's factor, fibronectin and plasminogen. The glue is made up of equal amounts of cryoprecipitate and venom fraction previously diluted in calcium chloride. The mixture of these components occurs only at the time of glue application on tissue during surgery.

SURGICAL PROCEDURE AND APPLICATION OF THE GLUE: In the present study, 30 healthy adult rams were used. They were divided into 3 groups of 10 animals each as follows: G1: fibrin glue group (application of fibrin glue on puncture sites and skin incisions after bilateral testicular biopsy with Tru-Cut needle) [Baxter Lab., Mortom Grove, III]; G2: swab/nylon group (hemostasis by compression with a swab on puncture sites and skin suturing with nylon after biopsy) and G3: control group (theanimals were not subjected either to biopsy or to surgery).

The rams were starved for 24 h. Then, their lumbosacral regions were clipped and rinsed with iodated alcohol for epidural anesthesia, using Lidocaine Chloridrate [Merrel Lepetit Farmacêutica e Ind. Ltda] 2.0 to 3.0 ml and 40/8 to 60/10 needles, depending on the animals' size and weight. The rams were kept on right lateral recumbency with the 4 limbs tied together and the anterior portion of their body kept raised. The scrotum was pulled caudally. The scrotum skin was prepared by standard surgical procedures, and the area was draped with a 12x6 cm-fenestrated drape. A latex rubber was placed on the bottom of the scrotum and held firmly to avoid a marked hemorrhage during surgery. The incision was made over the middle third of the posterior lateral surface of the testicle. The testicle was held firmly and a 2-cm incision was made with a scalpel blade longitudinally through the scrotal skin, dartos, scrotal fascia and tunica vaginalis parietal. Then, the testicle was slightly rotated following Larsen's recommendations (14) not to lay in apposition of the site of incision of the tunica vaginalis with that of the Tru-Cut needle into the visceral and albuginea tunics to reduce the incidence of adhesions. The Tru-Cut needle was inserted into the testicular parenchyma at 45 degrees, distal-proximallyand deeply enough to collect testicular fragments for histological examination. When the needle was withdrawn, hemorrhage was controlled by the local application of fibrin glue to animals of group 1 (Figure 1). In animals of group 2, gentle local mechanical compression with a sterile swab was performed (Figure 2). It took 2 to 3 min for both fibrin glue and compression with a swab to act. Right after bleeding control, skin was sealedwith fibrin glue. The margins of the surgical wound were held by a plastic "banana clip" and the glue was applied on the incision line (Figure 3). Three minutes later, the glue adhesive effect was achieved in animals of group 1. The animals of group 2 had their skin sutured with horizontal U-like stitches with 3-0 nylon [Point Suture do Brasil] (Figure 4). After surgery, the latex rubber was removed and the animals of both groups were given a 30,000 IU/Kg of body weight of a Penicillin antibiotic [Lab. WYETH LDA].

POSTOPERATIVE MONITORING: Postoperative monitoring consisted of daily observation of wound healing evolution until complete recovery from surgery. The stitches were removed 7 days after surgery in animals of group 2. On the 20 day after surgery, presence of adhesion strands was evaluated by palpation among the incision sites of the skin, tunics and testicle. One hundred days after surgery, bilateral orchiectomy was carried out and the material collected was macroscopically evaluated for the presence of subcutaneous (SC) adhesions or adhesion of the tunics.

RESULTS

ANESTHESIA: Local epidural anesthesia produced good analgesia, permitting manipulation of the testicles for up to 60 minutes.

LENGTH OF SURGERIES: Time elapsed from incision until suturing or sealing was of approximately 10 minutes for group 2 and 16 minutes for group 1.

ADHESIVE PROPERTY OF THE GLUE: The adhesive property of the glue was observed during immediate postoperative monitoring. The margins of the surgical wound were kept close together immediately after removal of the "banana clip", 3 min after application of the glue. Dehiscence of the wounds was not observed in suturing or sealingcarried out in all animals of both groups, despite the fact that no local dressing was used during the postoperative period.

ADHESION FORMATIONS: On the 20 day after surgery, adhesion strands were seen in 3 testicles (15%) in G1 and in 2 testicles (10%) in G2. In spite of not having been subjected to biopsy, G3 was examined and did not present any abnormality. One hundred days after surgery, G1 and G2 presented 4 testicles each (20%) with adhesions between the tunics at biopsy site. One of the testicles showed two adhesions between the tunics arising from two biopsy insertions having been made for ample sample collection (Figure 11).

Adhesions in SC were observed once (5%) in G1 and three times (15%) in G2, revealing a tendency of reduction of adhesion formation in SC of the scrotum by fibrin glue. There were a few adhesions observed,and these were generally discreet in both groups, except for one animal of G2 which had diffuse subcutaneous adhesions.

DISCUSSION

Fibrin glue is a biological sealant that imitates the last step of the normal coagulation process. In the presence of calcium ions, fibrinogen and factor XIII are activated by the "thrombin-like" enzyme. Fibrinogen is converted into fibrin and the fibrin molecules polymerize to form a stable fibrin clot that is strengthened by activated factor XIII. Fibronectin stimulates fibroblast proliferation that encourages the adhesion of fibroblasts to fibrin and collagen fibers(17,26).

Fibrin glue derived from snake venom used in testicular biopsypresentssome advantagesoverthe traditional method because surgical materials such as needle holders, suture lines, needles as well as hemostasis by mechanical compression are no longer needed.

In the present experiment, the remarkable adhesive property of FG for surgical wound closure was observed. Dehiscence of the woundswas not observed in both groups despite not having used postoperative wound dressing, differing from some authors such as Stolf et al.(22).

The organs involved in genito-urinary surgery are notable for their rich vascular supply and tendency to postoperative bleeding(17). For this reason, fibrin glue, which acts directly on hemorrhage control(13), plays an important role in testicular biopsies, since it helps prevent formation of hematomasbetween the tunicas vaginalis and the SC of the scrotum. These hematomas generally cause testicular degeneration or adhesion formation(18). The latter might compromise testicular mobility, and consequently, its thermoregulation(6). Ipso facto, one animal of G2 presented scrotal subcutaneous hematoma and developed testicular degeneration and diffuse subcutaneous adhesions.

With regard to adhesion formation, fibrin serves as a scaffold for fibroblast proliferation, which encourages collagen production. Thus, fibrin glue would increase adhesion formation. On the contrary, fibrin glue inhibits adhesion formation. Researchers who worked with experimental models of intra-abdominal adhesions in rats(5,17,21) proposed some hypotheses to explain this phenomenon. Since one of the causes of adhesion formation is the contact of two ischemic surfaces, the glue after coagulation might exert a mechanical action separating the above-mentioned surfaces. In addition, these researchers suggest that fibrin glue provides a chemical barrier, isolating the healing wound and its inflammatory mediating factors from the rest of thebody.

Concerning wound healing, fibrin is of vital importance because itstimulates the growth of fibroblasts, and the network formed acts both as a scaffold for migrating fibroblasts and as a hemostatic barrier(15). Thrombin was also found to have stimulatory and mitogenic effects on cell cultures of fibroblasts. Factor XIII has been reported to accelerate the wound healing process and demonstrated to be essential for normal fibroblast proliferation. However, Byrne et al.(2) reported that high concentrations of fibrinogen and thrombin inhibited the activity of neutrophils and macrophages, leading to the need for standardization of ideal quantities of the glue components.

In the present study, the use of the glue with adequate concentrations of its components yielded a faster and uniform healing process (according to descriptions of Iuan et al.(11) and Viterbo et al.(25)) with a better cosmetic appearance than traditional suturing, in addition to ruling out stitch removal.

Surgeries last, on average, 6 minutes more with the use of the gluedue to time spent in the formation of fibrin network between the skin margins.However, this additional time required for this technique does not pose any problem to the animal.

According to Byrne et al.(2), the main advantages of fibrin glue include tissue compatibility, biodegradability and efficacy when applied on wet surfaces or on those with rich vascular supply. The glue has no tissue toxicity and is reabsorbed after application, probably due to the action of plasminogen on fribrinolisis(17).

This was the first study carried out using fibrin glue derived from snake venom in rams after its successful use in rats, mice and rabbits. In addition, in the present study, homologous fibrinogen was used which might explain the apparent absence of any foreign body effect on the testicle or an anaphylatic reaction.

The use of fibrin glue in testicular biopsy of rams could be an important step to encourage its extensive and frequent use because the glue has several advantages that tend to reduce complications resulting from the surgical procedure.

ACKNOWLEDGEMENTS

The authors are very grateful to the Research Support Foundation of the State of São Paulo - FAPESP (n° 95/3958-0) for financing this study.

FIGURES 1 to 4: Use of fibrin glue (Figure 1) and swab (Figure 2) as the biopsy needle is withdrawn from the testicle. Application of fibrin glue on the margins of the surgical wound (Figure 3). Skin suturing with nylon (Figure 4).

FIGURES 5 to 8: Detail of the surgical wound 1 day (Figure 5) and 5 days (Figure 7) after fibrin glue application, and 1 day (Figure 6) and 7 days (Figure 8) after suturing with nylon.

FIGURES 9 and 10: Detail of the scar on scrotal skin of rams on the sites of application of fibrin glue derived from snake venom (Figure 9) and suturing with 3.0 nylon 100 days after surgery (Figure 10).

FIGURE 11: The right testicle of one ram of group 1 presenting adhesion formations between tunics at the sites of biopsy needle insertion.